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Characterization of the Blue–Light‐Activated Adenylyl Cyclase mPAC by Flash Photolysis and FTIR Spectroscopy
Author(s) -
Kerruth Silke,
Langner Pit,
Raffelberg Sarah,
Gärtner Wolfgang,
Heberle Joachim
Publication year - 2017
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.12746
Subject(s) - chemistry , fourier transform infrared spectroscopy , flash photolysis , photochemistry , adduct , adenylyl cyclase , protonation , spectroscopy , conformational isomerism , molecule , analytical chemistry (journal) , reaction rate constant , receptor , organic chemistry , ion , kinetics , physics , quantum mechanics , biochemistry
The recently discovered photo‐activated adenylyl cyclase ( mPAC from Microcoleus chthonoplastes) is the first PAC that owes a light‐, oxygen‐ and voltage‐sensitive ( LOV ) domain for blue‐light sensing. The photoreaction of the mPAC receptor was studied by time‐resolved UV /vis and light‐induced Fourier transform infrared ( FTIR ) absorption difference spectroscopy. The photocycle comprises of the typical triplet state LOV 715 and the thio‐adduct state LOV 390 . While the adduct state decays with a time constant of 8 s, the lifetime of the triplet state is with 656 ns significantly shorter than in all other reported LOV domains. The light‐induced FTIR difference spectrum shows the typical bands of the LOV 390 and LOV 450 intermediates. The negative S‐H stretching vibration at 2573 cm −1 is asymmetric suggesting two rotamer configurations of the protonated side chain of C194. A positive band at 3632 cm −1 is observed, which is assigned to an internal water molecule. In contrast to other LOV domains, mPAC exhibits a second positive feature at 3674 cm −1 which is due to the O‐H stretch of a second intrinsic water molecule and the side chain of Y476. We conclude that the latter might be involved in the dimerization of the cyclase domain which is crucial for ATP binding.